The objectives of this research are to determine the chemical mechanisms of the concerted interactions of several intermediary metabolites with an allosteric enzyme, and the relationship of the structure of these enzymes to their function both in vitro and in vivo. The enzyme chosen for these studies is the phosphoenolpyruvate carboxylase of Escherichia coli. This is an allosteric enzyme that plays a major role in the control of flow of intermediary metabolite@ into the citric acid cycle. Some of the compounds that stimulate its catalytic activity in vitro are acetyl coenzyme A, some nucleotide di-and triphosphates, long chain fatty acids and their coenzyme A derivatives, fructose-1, 6-bisphosphate, and some organic solvents. The most effective negative modifiers of this enzyme are four-carbon dicarboxylic acids that contain a nucleophilic group in the L-alpha-configuration, e.g., L-aspartate, and L-malate. The mechanism of the interactions of these effectors on the enzyme, singly and in combinations, are studied in vitro and in situ primarily by kinetic methods, but augmented by other physical and chemical methods where feasible. Enzymes with alterations in either their catalytic or regulatory functions are isolated and used to determine which structural changes have occurred and how they relate to the altered functions. Conventional methods of amino acid and peptide analyses are used for determining structural changes.